Herpes simplex virus 2 (HSV-2) causes genital herpes and increases the risk of transmission and infection with HIV. Thus a vaccine for HSV-2 would not only reduce the rate of genital herpes, but also might reduce spread of HIV. Several HSV-2 vaccines have been tested in humans for prevention or reduction of genital herpes disease, but none has been licensed for use in humans. Last year we initiated a phase 1 clinical trial to test the safety and immunogenicity of a different vaccine, a replication-defective HSV-2 vaccine, in humans. This vaccine was developed by Dr. David Knipe at Harvard, and our lab performed much of the preclinical work in mice and guinea pigs. This vaccine trial is continuing this year and we have fully enrolled the study. Patients are continuing to be vaccinated. Last year we also tested the ability of interleukin-1 (IL-1) to serve as an adjuvant to enhance the effectiveness of an HSV-2 gD vaccine. We found that mice receiving HSV-2 gD with IL-1 combined with alum and monophosphoryl lipid A(MPL) adjuvant had improved survival after challenge with HSV-2 compared with animals receiving gD with alum and MPL. HSV-2 glycoproteins B (gB) and D (gD) are targets of neutralizing antibodies and T cells, but clinical trials involving intramuscular (i.m.) injection of HSV-2 gB and gD in adjuvants have not been effective. This year we evaluated intravaginal (ivag) genetic immunization of mice with a replication-defective human papillomavirus pseudovirus expressing HSV-2 gB (HPV-gB) or gD (HPV-gD) constructs to target different subcellular compartments. The HPV vaccines consists of a single viral protein that self assembles to form a virus-like particle that induces potent antibody production and protects against infection with HPV. HPV pseudoviruses contain an HPV protein shell which surrounds a DNA expression vector that can express foreign proteins. Thus, these pseudoviruses transduce recipient cells with the plasmid and the cells express the foreign protein. HPV pseudoviruses expressing a secreted form of HSV-2 gB (gBsec) or gD (gDsec), but not HPV pseudoviruses expressing a cytoplasmic or membrane-bound form, induced circulating and intravaginal-tissue-resident memory CD8 T lymphocytes that were able to secrete gamma interferon and tumor necrosis factor alpha as well as moderate levels of serum HSV neutralizing antibodies. Combined immunization with HPV pseudoviruses expressing gBsec and gDsec (HPV-gBsec/gDsec) vaccines conferred longer survival after vaginal challenge with HSV-2 than immunization with HPV-gBsec or HPV-gDsec alone. HPV-gBsec/gDsec ivag vaccination was associated with a reduced severity of genital lesions and lower levels of viral shedding in the genital tract after HSV-2 challenge. In contrast, intramuscular vaccination with a soluble truncated gD protein (gD2t) in alum and monophosphoryl lipid A (MPL) elicited high neutralizing antibody titers and improved survival but did not reduce genital lesions and viral shedding. Vaccination combining ivag HPV-gBsec/gDsec and i.m. gD2t-alum-MPL improved survival and reduced genital lesions and viral shedding. Finally, high levels of circulating HSV-2-specific CD8 T lymphocytes, but not serum antibodies, correlated with reduced viral shedding. Taken together, our data underscore the potential of HPV pseudoviruses as a platform for a topical mucosal vaccine to control local manifestations of primary HSV-2 infection.